Sex Steroids and the Free Hormone Hypothesis

In their research article published in the September 9, 2005 issue of Cell, Hammes et al. (2005) investigated the role of megalin (an endocytic receptor in a variety of tissues) and SHBG (sex hormone binding globulin) on the cellular uptake of sex steroid hormones in mice. In plasma, androgens and estrogens can circulate bound tightly to SHBG, bound loosely to albumin, unbound, or free. Although oversimplified, the currently favored view—called the free hormone hypothesis—postulates that it is the free and albumin bound forms of sex steroid hormones that are available for diffusion out of capillaries and into cells, where they initiate an appropriate response. Hammes et al. (2005) report that megalin enables, and is crucial for, the internalization of sex steroids bound to SHBG, suggesting an alternative pathway for the uptake of sex steroid hormones. However, there are several important points that raise questions about the conclusions drawn from this study. Hammes et al. (2005) used both an in vitro model, the BN16 cell line that expresses megalin, and an in vivo model, the megalin knockout mouse (Nykjaer et al., 1999), to examine the interactions among megalin, SHBG, and certain sex steroids. Mice lacking megalin are severely deficient in vitamin D, and this deficiency has been reported to be associated with impaired gonadal development and function (Kinuta et al., 2000). Thus, it is possible that it is a deficiency in vitamin D and not a lack of megalin that causes the reproductive abnormalities observed by Hammes et al. in their megalin knockout mice. Furthermore, the adult mouse liver does not synthesize SHBG and there is no SHBG in adult mouse plasma (Janne et al., 1999). Thus, findings related to the importance of SHBG in the uptake of steroids and in steroid hormone action in this mouse model are unlikely to yield biologically relevant results. In addition, whereas the BN16 cell line expresses megalin, it does not express receptors for androgens and estrogens, calling into question the physiological relevance of studies on the uptake of steroid hormones in these cells. There are several other concerns regarding this study. The authors analyzed the specificity of the megalinSHBG interaction both before and after denaturation of megalin (see Hammes et al., 2005, Figure 1A). Although SHBG does not bind to denatured megalin, the binding of megalin to denatured SHBG was not investigated. It is feasible that megalin, which is known to bind to a large variety of protein and nonprotein ligands (Christensen and Birn, 2002), may be able to bind biologically inactive 125I-SHBG resulting in a misinterpretation of these experiments. The authors ascertained the uptake of 3H-testosterone by BN16 cells from media containing SHBG (Figure 1C), but only after a 5 hr incubation. They did not chemically characterize the molecules that had incorporated the 3H-label—it could have been incorporated into any number of metabolites of testosterone. In the absence of kinetic data, it is not clear whether other events confounded the interpretation of the results, such as rapid uptake of testosterone with its subsequent release into the medium. Testosterone is known to rapidly enter cells in vitro and to be metabolized extensively; the metabolites exist in cells and are released into the medium (Smith et al., 1994). When Hammes et al. (2005) incubated BN16 cells with a 5-fold excess of SHBG (500 nM) relative to 3H-testosterone, only 11% of the 3H-label was associated with the cells (Figure 1C). Furthermore, the 3H-compounds were not identified. If the suggestion by Hammes et al.—that sex steroid hormones bound to SHBG are taken up by cells with the help of megalin—were correct, then one would expect more than 11% uptake of the bound steroid. At zero time, essentially all of the 3H-testosterone is bound; thus the steroid uptake should have been roughly equal to the 40% uptake for 125I-